The susceptibility of colorectal carcinomas(CRC) to active specific immunotherapy is suggested by several clinical trials with whole tumor cell or anti-idotypic antibody vaccines. With the availability of cloned tumor antigens, new potentially improved approaches to active immunotherapy have become available. These antigens are more specific than tumor cell vaccines and most likely more effective than anti-idiotypes. This program project focuses on the CRC-associated antigen CO17- 1A/GA733(referred to as GA733) which has shown great promise in passive and active immunotherapy of CRC patients over the past 15 years. The overall hypothesis that immunizations of patients with the GA733 antigen and mice with the murine homolog of the GA733 antigen [murine epithelial glycoprotein (mEGP)], both expressed in recombinant adenoviruses (Ad), can break immunologic tolerance to the antigen expressed by both tumor and normal tissues, will be tested (Projects 1-4). The mechanism(s) of vaccine effects will be investigated in all four projects using both immunological and structural/functional approaches. As the result of the studies proposed in Projects 1, 2 and 4, modified GA733 antigen vaccines with immunotherapeutic potential may be identified with utility for future clinical trials. All areas of research proposed here are highly interrelated and each project contributes to and benefits from the other projects. The goal of Project 1 is to study the molecular basis of GA733 antigen function in CRC and normal cells by investigating structural and functional properties of this protein. A better understanding of the structural and functional role of the GA733 antigen will provide insight into the mechanisms for potential anti-tumor effects and toxicities of AdGA733-2 and mEGP vaccines. A major goal of Project is to investigate the mechanism underlying anti-tumor effects of AdmEGP vaccine already demonstrated in mice and to evaluate modified vaccination strategies as a basis for future clinical trials with AdGA733-2 vaccine. In Project 3, a phase I clinical trial with AdGA733-2 is proposed with the primary goal to determine immunogenicity and toxicity of the vaccine. A follow-up phase I trial will build upon the results obtained in the mouse model in Project 2 and will likely include the combined administration of AdGA733-2 and IL- 2. The goal of Project 4 is to evaluate humoral and cellular immune responses to the GA733 antigen may provide novel vaccines with improved immunotherapeutic potential for future clinical trials. It is expected that this program will contribute to improvement and better understanding of active immunotherapy against CRC.